Complex transcription regulation of acidic chitinase suggests fine-tuning of digestive processes in Drosera binata.

Abstract

Main conclusion: DbChitI-3, Drosera binata's acidic chitinase, peaks at pH 2.5 from 15 °C to 30 °C. Gene expression is stimulated by polysaccharides and suppressed by monosaccharide digestion, implying a feedback loop in its transcriptional regulation. Here, we characterised a novel chitinase gene (DbChitI-3) isolated from the carnivorous plant species Drosera binata with strong homology to other Drosera species' extracellular class I chitinases with a role in digestive processes. The capability to cleave different forms of chitin was tested using recombinantly produced chitinase in Escherichia coli (rDbChitI-3^S-His) and subsequent purification. The recombinant protein did not cleave chitin powder, the mono-, di- and tri- N-acetyl-D-glucosamine substrates, but cleaved acetic acid-swollen chitin. Fluorometric assay with acetic acid-swollen FITC-chitin as a substrate revealed the maximum enzyme activity at pH 2.5, spanning from 15 °C to 30 °C. Comparing enzymatic parameters with commercial chitinase from Streptomyces griseus showed rDbChitI-3S-His efficiency reaching 64.3% of S. griseus chitinase under optimal conditions. The highest basal expression of DbChitI-3 was detected in leaf blades. In other organs, the expression was either fivefold lower (petioles) or almost nondetectable (stems, roots and flowers). Application of gelatin, chitin, and pachyman resulted in a 3.9-, 4.6- and 5.7-fold increase in the mRNA transcript abundance of DbChitI-3 in leaves. In contrast, monosaccharides and laminarin decreased transcription of the DbChitI-3 gene by at least 70%, 5 h after treatment. The simultaneous application of suppressor and inducer (glucose and pachyman) indicated the predominant effect of the suppressor, implying that sufficient monosaccharide nutrients prioritize absorption processes in D. binata leaves over further digestion of the potential substrate.